Bob Fuller

Personal Information

My lab takes a comprehensive approach to studying biological problems, utilizing methods of genetics, molecular and cell biology and biochemistry to understand cellular events at the molecular level within a physiological context. Students in the lab typically learn to use multiple approaches in their projects to address the core interests of the lab in protein processing and localization in the secretory/endocytic pathways.

Proteolytic Processing and Protein Localization in the Secretory Pathway in Yeast and Mammals. A key event in the biosynthesis of many bioactive peptides and secretory and transmembrane proteins is proteolytic maturation of proprotein precursors by cleavage at clusters of Lys and Arg residues. Such reactions are catalyzed by proteases in late secretory compartments, such as the trans Golgi network (TGN) and secretory granules, and at the plasma membrane. Studies of proprotein processing in the yeast Saccharomyces cerevisiae led to the discovery of the functionally diverse kexin/furin family of enzymes responsible for such processing reactions in eukaryotes from yeast to humans. We are interested in understanding both the biochemical and cellular determinants of specificity and efficiency in proprotein processing.

Protein localization in the Yeast TGN-endosomal system. Processing of secretory proteins is compartmentalized, requiring colocalization of substrates and processing enzymes. We use yeast as a system in which to study how proteins are localized to specific secretory compartments in eukaryotic cells. We have identified several localization signals in the C-terminal cytosolic tail of Kex2 protease and have shown that steady-state localization of Kex2p to the TGN depends upon continued cycles of vesicular transport of the protein between the TGN and endosomal compartments. Using genetic approaches, we have identified novel proteins required for specific vesicle-mediated transport steps in TGN-endosomal trafficking. Soi1/Vps13p is a highly conserved protein required for retrograde transport from the late endosome to the TGN. Two SOI1 homologues have recently been identified as loci for two complex human genetic diseases Chorea-Acanthocytosis and Cohen Syndrome. Soi3/Rav1p, also a conserved protein, functions at the early endosome in regulated activation/assemply of proton-translocating Vacuolar ATPase and is essential for early endosome to late endosome trafficking.

To dissect molecular events in vesicular transport in the TGN-endosomal system, we have developed a cell-free assay for vesicular transport between the TGN and the late endosome, a major sorting event that separates lysosomal/vacuolar cargo from secreted proteins. This reaction requires factors involved in formation (clathrin, the dynamin homologue Vps1, Gga1/2 clathrin adaptors) and consumption (the late endosomal syntaxin Pep12p, Vps21P & Vps45p) of clathrin-coated vesicles. We are exploiting this assay to identify molecular mechanisms of cargo sorting and vesicle formation at the TGN and targeting and fusion of transport carriers to the late endosome. In collaboration with Adam Hoppe of Microbiology and Immunology and Anuj Kumar in Molecular Cellular and Developmental Biology, we are applying fluorescence resonance energy transfer microscopy to study events in vesicular transport in living yeast cells.

Proteolytic Processing. At the basic biochemical level, we use the yeast Kex2 protease to understand structure-function relationships in this family of enzymes. The mammalian enzymes, typified by the widely expressed human enzyme furin, process not only a wide array of physiological substrates (including growth factors, receptors, clotting factors, metallo and aspartyl proteases and matrix proteins) but also activate important pathophysiological substrates that include the envelope glycoproteins of important viral pathogens (HIV-1, Ebola Virus, Avian Influenza) and toxin components of important bacterial pathogens (Anthrax, Diptheria, Pseudomonas). Furin-family enzymes are also implicated in tumor metastasis and osteoarthritis. We are developing furin-family inhibitors using both small molecule and protein engineering approaches to provide novel drug models for infectious and chronic diseases.